Use of the cd2 signaling domain in second-generation chimeric antigen receptors

ABSTRACT

The present invention provides compositions and methods for treating cancer in a human. The invention includes relates to administering a genetically modified T cell expressing a CAR having an antigen binding domain, a transmembrane domain, a CD2 signaling domain, and a CD3 zeta signaling domain. The invention also includes incorporating CD2 into the CAR to alter the cytokine production of CAR-T cells in both negative and positive directions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 61/601,907, filed Feb. 22, 2012, the contents of which areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

T lymphocytes modified with chimeric-antigen receptors (CARs) bearingthe CD3-zeta signaling domain exhibit moderate levels of target tumorcytolysis and low amounts of cytokine production. The incorporation ofcostimulatory signaling domains, usually that of CD28 or 4-1BB, intosecond-generation CARs significantly increases target cell killing andcytokine production. It has been shown that ligation of the natural CD2molecule resulted in quantitative and qualitative differences comparedto TCR-induced calcium signal transduction. Recent reports demonstratedthat ligation of the endogenous CD2 receptor significantly increasesIL-2 produced from first-generation CAR-T cells.

Current data indicates that CAR T cells need to engraft and survive inpatients who have clinically beneficial tumor responses (Porter et al.,2011, N Engl J Med, 365:725-33). However, activation-induced T celldeath can occur when the threshold for too much signal transduction isreached (Viola et al., 1996, Science. 273(5271):104-6; Ren-Heidenreichet al., 2002, Cancer Immunol Immunother, 2002:417-23; Bachmann et al.,1996, Eur J Immunol, 26(9):2017-22; Ren-Heidenreich et al., 2001, CurrGene Ther, 1(3):253-5; Borthwick et al., 1996, Immunology, (4):508-15).

Thus, there is an urgent need in the art for compositions of CARs thatregulate T cell responses to enhance CAR T cell survival and provide abeneficial environment for anti-tumor activity. The present inventionaddresses this need.

SUMMARY OF THE INVENTION

The invention provides an isolated nucleic acid sequence encoding achimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain, a transmembrane domain, a costimulatory signalingregion, and a CD3 zeta signaling domain, wherein the costimulatorysignaling region comprises the CD2 signaling domain.

In one embodiment, the CAR comprises the nucleic acid sequence of SEQ IDNO: 1.

In one embodiment, the antigen binding domain is an antibody or anantigen-binding fragment thereof. In one embodiment the antigen-bindingfragment is a Fab or a scFv. In one embodiment, the antigen bindingdomain binds to a tumor antigen. In one embodiment the tumor antigen isassociated with a hematologic malignancy. In one embodiment, the tumorantigen is associated with a solid tumor. In one embodiment, tumorantigen is selected from the group consisting of CD19, CD20, CD22, ROR1,mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2,NY-ESO-1 TCR, MAGE A3 TCR, and any combination thereof.

In one embodiment, the costimulatory signaling region further comprisesthe intracellular domain of a costimulatory molecule selected from thegroup consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C,B7-H3, a ligand that specifically binds with CD83, and any combinationthereof.

The invention also provides an isolated chimeric antigen receptor (CAR)comprising an antigen binding domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain, whereinthe costimulatory signaling region comprises the CD2 signaling domain.

The invention also provides a vector comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises anantigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein thecostimulatory signaling region comprises the CD2 signaling domain.

The invention also provides a cell comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), the CAR comprising anantigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein thecostimulatory signaling domain comprises the CD2 signaling domain.

In one embodiment, the cell is selected from the group consisting of a Tcell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and aregulatory T cell.

In one embodiment, the cell exhibits an anti-tumor immunity when theantigen binding domain binds to its corresponding antigen.

The invention also provides a method for stimulating a T cell-mediatedimmune response to a target cell population or tissue in a mammal, themethod comprising administering to a mammal an effective amount of acell genetically modified to express a CAR, wherein the CAR comprises anantigen binding domain, a transmembrane domain, a costimulatorysignaling region comprising the CD2 signaling domain, and a CD3 zetasignaling domain, and wherein the antigen binding domain is selected tospecifically recognize the target cell population or tissue.

The invention provides a method of providing an anti-tumor immunity in amammal, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a CAR, wherein the CARcomprises an antigen binding domain, a transmembrane domain, acostimulatory signaling region comprising the CD2 signaling domain, anda CD3 zeta signaling, thereby providing an anti-tumor immunity in themammal.

The invention provides a method of treating a mammal having a disease,disorder or condition associated with an elevated expression of a tumorantigen, the method comprising administering to the mammal an effectiveamount of a cell genetically modified to express a CAR, wherein the CARcomprises an antigen binding domain a transmembrane domain, acostimulatory signaling region comprising the CD2 signaling domain, anda CD3 zeta signaling domain, thereby treating the mammal.

In one embodiment, the cell is an autologous T cell.

The invention provides a method of treating a human with cancer, themethod comprising administering to the human a T cell geneticallyengineered to express a CAR, wherein the CAR comprises an antigenbinding domain, a transmembrane domain, a costimulatory signaling regioncomprising the CD2 signaling domain, and a CD3 zeta signaling domain.

In one embodiment, the human is resistant to at least onechemotherapeutic agent.

The invention provides a method of generating a persisting population ofgenetically engineered T cells in a human diagnosed with cancer, themethod comprising administering to the human a T cell geneticallyengineered to express a CAR, wherein the CAR comprises an antigenbinding domain a transmembrane domain, a costimulatory signaling regioncomprising the CD2 signaling domain, and a CD3 zeta signaling domain,wherein the persisting population of genetically engineered T cellspersists in the human for at least one month after administration.

In one embodiment, the persisting population of genetically engineered Tcells comprises at least one cell selected from the group consisting ofa T cell that was administered to the human, a progeny of a T cell thatwas administered to the human, and a combination thereof.

In one embodiment, the persisting population of genetically engineered Tcells comprises a memory T cell.

In one embodiment, the cancer is chronic lymphocytic leukemia. In oneembodiment, the chronic lymphocytic leukemia is refractory CD19+leukemia and lymphoma.

In one embodiment, the persisting population of genetically engineered Tcells persists in the human for at least three months afteradministration.

In one embodiment, the persisting population of genetically engineered Tcells persists in the human for at least four months, five months, sixmonths, seven months, eight months, nine months, ten months, elevenmonths, twelve months, two years, or three years after administration.

In one embodiment, the cancer is treated.

The invention provides a method of expanding a population of geneticallyengineered T cells in a human diagnosed with cancer, the methodcomprising administering to the human a T cell genetically engineered toexpress a CAR, wherein the CAR comprises an antigen binding domain, atransmembrane domain, a costimulatory signaling region comprising theCD2 signaling domain, and a CD3 zeta signaling domain, wherein theadministered genetically engineered T cell produces a population ofprogeny T cells in the human.

In one embodiment, the progeny T cells in the human comprise a memory Tcell.

In one embodiment, the T cell is an autologous T cell.

In one embodiment, the human is resistant to at least onechemotherapeutic agent.

The invention includes a method of modulating the amount of cytokinesecreted by a T cell, said method comprising genetically engineering theT cell to express a CAR, wherein the CAR comprises an antigen bindingdomain, a transmembrane domain, a costimulatory domain region comprisingCD2, and a CD3 zeta signaling domain.

In one embodiment, modulating the amount of cytokine secreted by a Tcell reduces the proliferation of T regulatory cells.

The invention includes a method of reducing the amount ofactivation-induced calcium influx into a T cell, said method comprisinggenetically engineering the T cell to express a CAR, wherein the CARcomprises an antigen binding domain, a transmembrane domain, acostimulatory domain region comprising CD2, and a CD3 zeta signalingdomain.

In one embodiment, reducing the amount of activation-induced calciuminflux into a T cell prevents the activation-induced cell death of the Tcell.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 depicts the nucleotide sequence for the antiCD19-CD2zeta CAR (SEQID NO: 1).

FIG. 2 depicts the results of example experiments demonstrating that Tcells activate and proliferate from CD3/CD2 signals. Normal Human donorCD4 and CD8 T cells were stimulated with magnetic beads coated with αCD2(T11.1 and T11.2), αCD3 (OKT3), and αCD28 (9.3) antibodies. Absolutecell number and population doublings demonstrate cell proliferation.Cell size represents state of cell activation. Activation by CD2/CD3signals sufficiently induces cell activation and proliferation.

FIG. 3 depicts the results of example experiments demonstrating that CD2CARs release cytokines characteristic of effective immune response. mRNACAR-electroporated normal human donor T cells were co-cultured for 24hours with target tumor cells and cytokine secretion was measured. IL-2and IFN-γ is increased in SS1CD2z, similar to SS128z (top). However, CD2signal diminishes cytokine secretion in SS128CD2z. CD19CD2z CAR producessimilar amounts IL-2 and IFN-g compared to CD19z and CD1928z.

FIG. 4 depicts the results of example experiments demonstrating thatCARs with the CD2 signaling domain are effective at killing tumor cells.mRNA CAR-electroporated normal human donor T cells were co-cultured withtarget tumor cells for 18 hours (left) and 4 hours (right) at varyingeffector:target ratios. CD2 CARs efficiently eliminate tumor cells inboth assays, comparable CARs with CD3z or CD28z signaling domains.

FIG. 5 depicts the results of an example experiment demonstrating thatCD2 signaling domain improves T-cell proliferation whenCAR-electroporated normal human donor T cells were co-cultured withtarget tumor cells for 5 days. There is no significant difference in thenumber of CD2 CAR-T cells at 3 days of stimulation, but there is atwo-fold increase in the number of CD2 CAR-T cells after 5 days,suggesting that CD2 signaling domain contributes to increased T-cellproliferation when added to a CAR.

FIG. 6 depicts the results of an example experiment demonstrating thecalcium flux response of CAR-T cells with the CD28, CD2, CD137 signalingdomains in addition to the CD3zeta domain. mRNA CAR-electroporatednormal human donor T cells were loaded with Indo-1 AM calcium indicatorand stimulated with mesothelin-Fc fusion protein 30 seconds after thestart of recording. Shown are the ratios of bound calcium (Indo-1violet) to free calcium (Indo-1 blue) over time. Additions of the CD2signaling domain to SS1z increases the calcium flux signal, but not asstrong as the SS128z calcium flux.

DETAILED DESCRIPTION

The invention relates to compositions and methods for treating cancer,including, but not limited, to hematologic malignancies and solidtumors. The present invention relates to a strategy of adoptive celltransfer of T cells modified to express a chimeric antigen receptor(CAR). CARs are molecules that combine antibody-based specificity for adesired antigen (e.g., tumor antigen) with a T cell receptor-activatingintracellular domain to generate a chimeric protein that exhibits aspecific anti-tumor cellular immune activity.

The present invention provides for the incorporation of CD2 into a CARin order to alter the cytokine production of T cells that are engineeredto express the CAR comprising CD2. As discussed elsewhere herein, CD2can be incorporated into a CAR to both negatively and positivelyregulate cytokine production of T cells. Therefore, the use of CD2 in aCAR can alter CAR T cell survival and activation induced cell deaththresholds.

In one embodiment, the invention provides a method of modulating theamount of cytokine secreted by a T cell. The method comprisesgenetically engineering the T cell to express a CAR wherein the CARcomprises an antigen binding domain, a transmembrane domain, acostimulatory domain region comprising CD2, and a CD3 zeta signalingdomain.

In another embodiment, the invention provides a method of reducing theamount of activation-induced calcium influx into a T cell. The methodcomprises genetically engineering the T cell to express a CAR, whereinthe CAR comprises an antigen binding domain, a transmembrane domain, acostimulatory domain region comprising CD2, and a CD3 zeta signalingdomain.

The present invention relates generally to the use of T cellsgenetically modified to stably express a desired CAR. T cells expressinga CAR are referred to herein as CAR T cells or CAR modified T cells.Preferably, the cell can be genetically modified to stably express anantibody binding domain on its surface, conferring novel antigenspecificity that is MHC independent. In some instances, the T cell isgenetically modified to stably express a CAR that combines an antigenrecognition domain of a specific antibody with an intracellular domainof the CD3-zeta chain or FcγRI protein into a single chimeric protein.

In one embodiment, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain, a transmembrane domain, anda cytoplasmic domain. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Inanother embodiment, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.Preferably, the transmembrane domain is the CD8α hinge domain.

With respect to the cytoplasmic domain, the CAR of the invention can bedesigned to comprise the CD2, CD28 and/or 4-1BB signaling domain byitself or be combined with any other desired cytoplasmic domain(s)useful in the context of the CAR of the invention. In one embodiment,the cytoplasmic domain of the CAR can be designed to further comprisethe signaling domain of CD3-zeta. For example, the cytoplasmic domain ofthe CAR can include, but is not limited to, CD3-zeta, CD2, 4-1BB andCD28 signaling modules, and combinations thereof. Accordingly, theinvention provides CAR T cells and methods of their use for adoptivetherapy.

In one embodiment, the CAR T cells of the invention can be generated byintroducing a lentiviral vector comprising the desired CAR, for examplea CAR comprising anti-CD19, CD8α hinge and transmembrane domain, andhuman CD2 and CD3zeta signaling domains, into the cells. In oneembodiment, the CART cells of the invention are able to replicate invivo resulting in long-term persistence that can lead to sustained tumorcontrol.

In another embodiment, the CAR T cells of the invention can be generatedby transfecting an RNA encoding the desired CAR, for example a CARcomprising anti-CD19, CD8α hinge and transmembrane domain, and human CD2and CD3-zeta signaling domains, into the cells. In one embodiment, theCAR is transiently expressed in the genetically modified CAR T cells.

In one embodiment the invention relates to administering a geneticallymodified T cell expressing a CAR for the treatment of a patient havingcancer or at risk of having cancer using lymphocyte infusion.Preferably, autologous lymphocyte infusion is used in the treatment.Autologous PBMCs are collected from a patient in need of treatment and Tcells are activated and expanded using the methods described herein andknown in the art and then infused back into the patient.

In one embodiment, the invention relates to genetically modified T cellsexpressing a CAR for the treatment of a patient with cancer. The presentinvention is based upon the finding that the inclusion of the CD2signaling domain within the cytoplasmic domain of a CAR significantlyinfluences cytokine production and calcium signaling in CAR modified Tcells. In one embodiment, inclusion of the CD2 signaling domainincreases cytokine production. In another embodiment, inclusion of theCD2 signaling domain reduces cytokine production. Therefore, the CD2signaling domain within the cytoplasmic domain of a CAR, either alone orin combination with other cytoplasmic signaling domains, positively andnegatively regulates cytokine production at the tumor site. In anotherembodiment, the CD2 signaling domain within a CAR reduces calciuminflux, thereby preventing activation-induced cell death of thegenetically modified CAR.

In yet another embodiment, the invention relates generally to thetreatment of a patient at risk of developing cancer. The invention alsoincludes treating a malignancy or an autoimmune disease in whichchemotherapy and/or immunotherapy in a patient results in significantimmunosuppression in the patient, thereby increasing the risk of thepatient of developing cancer.

The invention includes using T cells expressing an anti-CD19 CAR,including both CD3-zeta and the CD2 costimulatory domain (also referredto as CART19 T cells). In one embodiment, the CART19 T cells of theinvention can undergo robust in vivo T cell expansion and can establishCD19-specific memory cells that persist at high levels for an extendedamount of time in blood and bone marrow. In some instances, the CART19 Tcells of the invention infused into a patient can eliminate leukemiacells in vivo in patients with advanced chemotherapy-resistant CLL.However, the invention is not limited to CART19 T cells. Rather, theinvention includes any antigen binding domain (e.g. anti-mesothelin)fused with one or more intracellular domains selected from the group ofa CD2 signaling domain, a CD137 (4-1BB) signaling domain, a CD28signaling domain, a CD3zeta signal domain, and any combination thereof.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared ×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention provides compositions and methods for treatingcancer, as well as other diseases. The cancer may be a hematologicalmalignancy, a solid tumor, a primary or a metastasizing tumor. Otherdiseases treatable using the compositions and methods of the inventioninclude viral, bacterial and parasitic infections as well as autoimmunediseases.

In one embodiment, the invention provides a cell (e.g., T cell)engineered to express a CAR, wherein the CAR T cell exhibits anantitumor property. The CAR of the invention can be engineered tocomprise an extracellular domain having an antigen binding domain fusedto an intracellular signaling domain of the T cell antigen receptorcomplex zeta chain (e.g., CD3-zeta). The CAR of the invention whenexpressed in a T cell is able to redirect antigen recognition based onthe antigen binding specificity. An exemplary antigen is CD19 becausethis antigen is expressed on malignant B cells. However, the inventionis not limited to targeting CD 19. Rather, the invention includes anyantigen binding domain that when bound to its cognate antigen, affects atumor cell so that the tumor cell fails to grow, is prompted to die, orotherwise is affected so that the tumor burden in a patient isdiminished or eliminated. The antigen binding domain is preferably fusedwith an intracellular domain from one or more of a costimulatorymolecule and a zeta chain. Preferably, the antigen binding domain isfused with one or more intracellular domains selected from the group ofa CD2 signaling domain, CD137 (4-1BB) signaling domain, a CD28 signalingdomain, a CD3zeta signal domain, and any combination thereof.

In one embodiment, the CAR of the invention comprises a CD2 signalingdomain. This is because the present invention is partly based on thediscovery that CAR-mediated T-cell responses can be further enhancedwith the addition of costimulatory domains. For example, inclusion ofthe CD2 signaling domain significantly influenced cytokine production inboth positive and negative directions, dependent on the presence andidentity of other costimulatory domains in the expressed CAR. In oneembodiment, the invention comprises a CAR comprising both CD2 and CD28domains. In one embodiment, a modified T cell expressing a CAR with boththe CD2 and CD28 domains releases significantly less IL-2 compared to Tcells expressing a CAR with CD28 but not CD2. In one embodiment, amodified T cell that releases less IL-2 reduces the proliferation ofimmunosuppressive Treg cells. Further, inclusion of the CD2 signalingdomain significantly reduced calcium influx. In one embodiment, thereduction of calcium influx by inclusion of CD2 reduces activationinduced CAR T cell death.

In some embodiments, the present invention is directed to a retroviralor lentiviral vector encoding a CAR that is stably integrated into a Tcell and stably expressed therein. In other embodiments, the presentinvention is directed to an RNA encoding CAR that is transfected into aT cell and transiently expressed therein. Transient, non-integratingexpression of CAR in a cell mitigates concerns associated with permanentand integrated expression of CAR in a cell.

Compositions

The present invention provides a chimeric antigen receptor (CAR)comprising an extracellular and intracellular domain. The extracellulardomain comprises a target-specific binding element otherwise referred toas an antigen binding domain. The intracellular domain or otherwise thecytoplasmic domain comprises, a costimulatory signaling region and azeta chain portion. The costimulatory signaling region refers to aportion of the CAR comprising the intracellular domain of acostimulatory molecule. Costimulatory molecules are cell surfacemolecules other than antigens receptors or their ligands that arerequired for an efficient response of lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ capand/or Internal Ribosome Entry Site (IRES), the gene to be expressed,and a polyA tail, typically 50-2000 bases in length. RNA so produced canefficiently transfect different kinds of cells. In one embodiment, thetemplate includes sequences for the CAR.

Preferably, the CAR comprises an extracellular domain, a transmembranedomain and a cytoplasmic domain. The extracellular domain andtransmembrane domain can be derived from any desired source of suchdomains.

Antigen Binding Domain

The extracellular domain may be obtained from any of the wide variety ofextracellular domains or secreted proteins associated with ligandbinding and/or signal transduction. In one embodiment, the extracellulardomain may consist of an Ig heavy chain which may in turn be covalentlyassociated with Ig light chain by virtue of the presence of CH1 andhinge regions, or may become covalently associated with other Igheavy/light chain complexes by virtue of the presence of hinge, CH2 andCH3 domains. In the latter case, the heavy/light chain complex thatbecomes joined to the chimeric construct may constitute an antibody witha specificity distinct from the antibody specificity of the chimericconstruct. Depending on the function of the antibody, the desiredstructure and the signal transduction, the entire chain may be used or atruncated chain may be used, where all or a part of the CH1, CH2, or CH3domains may be removed or all or part of the hinge region may beremoved.

The extracellular domain can be directed to any desired antigen. Forexample, when an antitumor CAR is desired, the extracellular domainchosen to be incorporated into the CAR can be an antigen that isassociated with the tumor. The tumor may be any type of tumor as long asit has a cell surface antigen which is recognized by the CAR. In anotherembodiment, the CAR may one for which a specific monoclonal antibodycurrently exists or can be generated in the future.

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenmoiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one embodiment, the retroviral or lentiviral vector comprisingcomprises a CAR designed to be directed to an antigen of interest by wayof engineering a desired antigen into the CAR. In the context of thepresent invention, “tumor antigen” or “hyperporoliferative disorderantigen” or “antigen associated with a hyperproliferative disorder”refer to antigens that are common to specific hyperproliferativedisorders. In certain aspects, the hyperproliferative disorder antigensof the present invention are derived from cancers including, but notlimited to, primary or metastatic melanoma, mesothelioma, thymoma,lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma,Hodgkins lymphoma, leukemias, uterine cancer, cervical cancer, bladdercancer, kidney cancer and adenocarcinomas such as breast cancer,prostate cancer, ovarian cancer, pancreatic cancer, and the like.

In another embodiment, the template for the RNA CAR is designed to bedirected to an antigen of interest by way of engineering a desiredantigen binding domain into the CAR. In the context of the presentinvention, “tumor antigen” or “hyperporoliferative disorder antigen” or“antigen associated with a hyperproliferative disorder” refer toantigens that are common to specific hyperproliferative disorders. Incertain aspects, the hyperproliferative disorder antigens of the presentinvention are derived from cancers including, but not limited to,primary or metastatic melanoma, mesothelioma, thymoma, lymphoma,sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkinslymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer,kidney cancer and adenocarcinomas such as breast cancer, prostatecancer, ovarian cancer, pancreatic cancer, and the like.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune responses. Inone embodiment, the tumor antigen of the present invention comprises oneor more antigenic cancer epitopes immunologically recognized by tumorinfiltrating lymphocytes (TIL) derived from a cancer tumor of a mammal.The selection of the antigen binding domain of the invention will dependon the particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogeneHER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetalantigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD19, CD20and CD37 areother candidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The type of tumor antigen referred to in the invention may also be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

In a preferred embodiment, the antigen binding domain portion of the CARtargets an antigen that includes but is not limited to CD19, CD20, CD22,ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII,GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen bindmoiety that is specific to the desired antigen target. For example, ifCD19 is the desired antigen that is to be targeted, an antibody for CD19can be used as the antigen bind moiety for incorporation into the CAR ofthe invention.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention is responsible for activation of at leastone of the normal effector functions of the immune cell in which the CARhas been placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the invention comprises a cytoplasmicsignaling sequence derived from CD3 zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR of the invention. For example, the cytoplasmic domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention in exemplified primarily with CD2 as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. Inanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD2. In yetanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28 and CD2.In yet another embodiment, the cytoplasmic domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28, CD2, and 4-1BB.

Vectors

The present invention encompasses a DNA construct comprising thesequence of a CAR, wherein the sequence comprises the nucleic acidsequence of an antigen binding domain operably linked to the nucleicacid sequence of an intracellular domain. An exemplary intracellulardomain that can be used in the CAR of the invention includes but is notlimited to the intracellular domain of CD3-zeta, CD28, 4-1BB, CD2 andthe like. In some instances, the CAR can comprise any combination ofCD3-zeta, CD28, 4-1BB, CD2 and the like.

In one embodiment, the CAR of the invention comprises anti-CD19 scFv,human CD8 hinge and transmembrane domain, and human CD2 and CD3zetasignaling domains. In one embodiment, the CAR of the invention comprisesthe nucleic acid sequence set forth in SEQ ID NO: 1. In anotherembodiment, the CAR of the invention comprises the amino acid sequenceencoded by the nucleic acid set forth in SEQ ID NO: 1.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

RNA Transfection

In one embodiment, the genetically modified T cells of the invention aremodified through the introduction of RNA. In one embodiment, an in vitrotranscribed RNA CAR can be introduced to a cell as a form of transienttransfection. The RNA is produced by in vitro transcription using apolymerase chain reaction (PCR)-generated template. DNA of interest fromany source can be directly converted by PCR into a template for in vitromRNA synthesis using appropriate primers and RNA polymerase. The sourceof the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA,cDNA, synthetic DNA sequence or any other appropriate source of DNA. Thedesired template for in vitro transcription is the CAR of the presentinvention. For example, the template for the RNA CAR comprises anextracellular domain comprising a single chain variable domain of ananti-tumor antibody; a transmembrane domain comprising the hinge andtransmembrane domain of CD8a; and a cytoplasmic domain comprises thesignaling domain of CD3-zeta and the signaling domain of CD2

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the DNA is a full length geneof interest of a portion of a gene. The gene can include some or all ofthe 5′ and/or 3′ untranslated regions (UTRs). The gene can include exonsand introns. In one embodiment, the DNA to be used for PCR is a humangene. In another embodiment, the DNA to be used for PCR is a human geneincluding the 5′ and 3′ UTRs. The DNA can alternatively be an artificialDNA sequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

Genes that can be used as sources of DNA for PCR include genes thatencode polypeptides that provide a therapeutic or prophylactic effect toan organism or that can be used to diagnose a disease or disorder in anorganism. Preferred genes are genes which are useful for a short termtreatment, or where there are safety concerns regarding dosage or theexpressed gene. For example, for treatment of cancer, autoimmunedisorders, parasitic, viral, bacterial, fungal or other infections, thetransgene(s) to be expressed may encode a polypeptide that functions asa ligand or receptor for cells of the immune system, or can function tostimulate or inhibit the immune system of an organism. In someembodiments, t is not desirable to have prolonged ongoing stimulation ofthe immune system, nor necessary to produce changes which last aftersuccessful treatment, since this may then elicit a new problem. Fortreatment of an autoimmune disorder, it may be desirable to inhibit orsuppress the immune system during a flare-up, but not long term, whichcould result in the patient becoming overly sensitive to an infection.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary”, as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between zero and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100 T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun, 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Genetically Modified T Cells

In some embodiments, the CAR sequences are delivered into cells using aretroviral or lentiviral vector. CAR-expressing retroviral andlentiviral vectors can be delivered into different types of eukaryoticcells as well as into tissues and whole organisms using transduced cellsas carriers or cell-free local or systemic delivery of encapsulated,bound or naked vectors. The method used can be for any purpose wherestable expression is required or sufficient.

In other embodiments, the CAR sequences are delivered into cells usingin vitro transcribed mRNA. In vitro transcribed mRNA CAR can bedelivered into different types of eukaryotic cells as well as intotissues and whole organisms using transfected cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedmRNA. The method used can be for any purpose where transient expressionis required or sufficient.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the chimeric receptor mRNAs with differentstructures and combination of their domains. For example, varying ofdifferent intracellular effector/costimulator domains on multiplechimeric receptors in the same cell allows determination of thestructure of the receptor combinations which assess the highest level ofcytotoxicity against multi-antigenic targets, and at the same timelowest cytotoxicity toward normal cells.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free: An RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely. Cloning of cells is not necessary because of the efficiencyof transfection of the RNA and its ability to uniformly modify theentire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation.Preferably, it is desirable to stabilize IVT-RNA using variousmodifications in order to achieve prolonged expression of transferredIVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct can be delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. No.6,678,556, U.S. Pat. No. 7,171,264, and U.S. Pat. No. 7,173,116.Apparatus for therapeutic application of electroporation are availablecommercially, e.g., the MedPulser™ DNA Electroporation Therapy System(Inovio/Genetronics, San Diego, Calif.), and are described in patentssuch as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No.5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S.Pat. No. 6,233,482; electroporation may also be used for transfection ofcells in vitro as described e.g. in US20070128708A1. Electroporation mayalso be utilized to deliver nucleic acids into cells in vitro.Accordingly, electroporation-mediated administration into cells ofnucleic acids including expression constructs utilizing any of the manyavailable devices and electroporation systems known to those of skill inthe art presents an exciting new means for delivering an RNA of interestto a target cell.

Sources of T Cells

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD 14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besançon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle:cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) modified toexpress a CAR that combines an antigen recognition domain of a specificantibody with an intracellular domain of CD3-zeta, CD28, 4-1BB, CD2, orany combinations thereof. Therefore, in some instances, the modified Tcell can elicit a CAR-mediated T-cell response.

The invention provides the use of a CAR to redirect the specificity of aprimary T cell to a tumor antigen. Thus, the present invention alsoprovides a method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal comprising the step ofadministering to the mammal a T cell that expresses a CAR, wherein theCAR comprises a binding moiety that specifically interacts with apredetermined target, a zeta chain portion comprising for example theintracellular domain of human CD3zeta, and a costimulatory signalingregion.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellis able to kill tumor cells in the recipient. Unlike antibody therapies,CAR T cells are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In one embodiment, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. For example, CAR T cells of theinvention can undergo robust in vivo T cell expansion and persist athigh levels for an extended amount of time in blood and bone marrow andform specific memory T cells. Without wishing to be bound by anyparticular theory, CAR T cells may differentiate in vivo into a centralmemory-like state upon encounter and subsequent elimination of targetcells expressing the surrogate antigen.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified T cells may be an activeor a passive immune response. In addition, the CAR mediated immuneresponse may be part of an adoptive immunotherapy approach in whichCAR-modified T cells induce an immune response specific to the antigenbinding domain in the CAR. For example, a CART19 cells elicits an immuneresponse specific against cells expressing CD19.

While the data disclosed herein specifically disclose mRNA comprisingeither anti-CD19 scFv derived from FMC63 murine monoclonal antibody orthe anti-mesothelin SS1 scFv, along with human CD8α hinge andtransmembrane domain, and human CD2 and CD3zeta signaling domains, theinvention should be construed to include any number of variations foreach of the components of the construct as described elsewhere herein.That is, the invention includes the use of any antigen binding domain inthe CAR to generate a CAR-mediated T-cell response specific to theantigen binding domain. For example, the antigen binding domain in theCAR of the invention can target a tumor antigen for the purposes oftreat cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the antigen bind moiety portion of the CAR of theinvention is designed to treat a particular cancer. For example, the CARdesigned to target CD19 can be used to treat cancers and disordersincluding but are not limited to pre-B ALL (pediatric indication), adultALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage postallogenic bone marrow transplantation, and the like.

In another embodiment, the CAR can be designed to target CD22 to treatdiffuse large B-cell lymphoma.

In one embodiment, cancers and disorders include but are not limited topre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma,diffuse large B-cell lymphoma, salvage post allogenic bone marrowtransplantation, and the like can be treated using a combination of CARsthat target CD19, CD20, CD22, and ROR1.

In one embodiment, the CAR can be designed to target mesothelin to treatmesothelioma, pancreatic cancer, ovarian cancer, and the like. Inanother embodiment, the CAR can be designed to target CD33/IL3Ra totreat acute myelogenous leukemia and the like. In a further embodiment,the CAR can be designed to target c-Met to treat triple negative breastcancer, non-small cell lung cancer, and the like.

In one embodiment, the CAR can be designed to target PSMA to treatprostate cancer and the like. In another embodiment, the CAR can bedesigned to target Glycolipid F77 to treat prostate cancer and the like.In a further embodiment, the CAR can be designed to target EGFRvIII totreat glioblastoma and the like.

In one embodiment, the CAR can be designed to target GD-2 to treatneuroblastoma, melanoma, and the like. In another embodiment, the CARcan be designed to target NY-ESO-1 TCR to treat myeloma, sarcoma,melanoma, and the like. In a further embodiment, the CAR can be designedto target MAGE A3 TCR to treat myeloma, sarcoma, melanoma, and the like.

However, the invention should not be construed to be limited to solelyto the antigen targets and diseases disclosed herein. Rather, theinvention should be construed to include any antigenic target that isassociated with a disease where a CAR can be used to treat the disease.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of cancer. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing cancer. Thus, the present inventionprovides methods for the treatment or prevention of cancer comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Use of the CD2 Signaling Domain in Second-Generation ChimericAntigen Receptors

The incorporation of costimulatory domains into chimeric antigenreceptors can increases cytokine production and target cell killing. CD2is a costimulatory molecule that may influence calcium-mediatedsignaling. It is herein examined whether inclusion of the CD2intracellular domain into CARs influences calcium influx/signaling andcytokine production.

The materials and methods employed in these experiments are nowdescribed.

CD2-Containing CARs

The CD2 costimulatory domain was cloned into various CD19 or mesothelinspecific CARs. For example, FIG. 1 displays the nucleotide sequence forthe antiCD19-CD2z CAR (SEQ ID NO: 1).

T Cells

Blood samples were obtained from the Human Immunology Core of theUniversity of Pennsylvania. Peripheral blood CD4⁺ and CD8⁺ T cells werenegatively isolated using RosetteSep Kits (Stem cell Technologies).Cells were cultured in RPMI 1640 media supplemented with 10% FCS,100-U/ml penicillin, 100 μg/ml streptomycin sulfate, 10 mM Hepes in a37° C. and 5% CO₂ incubator. For stimulation, CD4⁺ and CD8⁺ T cells werecultured with a combination of αCD2(T11.1 and T11.2), αCD3 (OKT3),and/or αCD28 (9.3) antibody coated magnetic beads.

mRNA Electroporation

Human T cells were electroporated with mRNA to express the CD28:CD3zetadomain, CD28:CD2:CD3zeta or the CD2:CD3zeta domains. In addition, CARswere prepared to express only the CD2, CD28 or CD3zeta domains. Donor Tcells were electroporated with CAR mRNA by the following methods. On day10 of culture, the magnetic bead activated T cells were collected andelectroporated. Two electroporation systems were used: BTX CM830(Harvard Apparatus BTX, Holliston, Mass., USA), and Maxcyte (MaxcyteInc, Rockville, Md., USA). For electroporation using BTX EM830, thestimulated T cells subjected to electroporation were washed three timeswith OPTI-MEM (Invitrogen) and resuspended in OPTI-MEM at the finalconcentration of 1−3×10⁸/ml. Subsequently, 0.1 to 0.2 ml of the cellswas mixed with 10 μg/0.1 ml T cells of IVT RNA (or as indicated) andelectroporated in a 2-mm cuvette (Harvard Apparatus BTX, Holliston,Mass., USA). For electroporation using Maxcyte, the instruction manualwas followed using OC-400 processing chamber (Maxcyte Inc, Rockville,Md., USA) with integrated programs. Expression of the CARs was verifiedby flow cytometry. Cells were washed and suspended in FACs buffer (PBSplus 0.1% sodium azide and 0.4% BSA). Biotin-labeled polyclonal goatanti-mouse F(ab)₂ antibodies (anti-Fab, Jackson Immunoresearch, WestGrove, Pa.) were added to the tube and the cells were incubated at 4° C.for 25 minutes and washed twice. The cells were then stained withphycoerythrin-labeled streptavidin (BD Pharmingen, San Diego, Calif.).

Cytokine Secretion

K562 target cells expressing mesothelin or CD19 were washed andsuspended at 10⁶ cells/mL in R10. One hundred thousand of each targetcell type were added to each of 2 wells of a 96 well round bottom plate(Corning). Effector CAR T cell cultures were washed and suspended at 10⁶cells/mL in R10. One hundred thousand effector T cells were combinedwith target cells in the indicated wells of the 96 well plate. Inaddition, wells containing T cells alone were prepared. The plates wereincubated at 37° C. for 18 to 20 hours. After the incubation,supernatant was harvested and subjected to an ELISA assay for IL-2 andIFN-gamma using standard methods (Pierce, Rockford, Ill.).

Specific Target Lysis

mRNA electroporated CAR T cells were co-cultured with various cancercell lines and analyzed by flow cytometry for specific target lysis.

Measuring Calcium Influx

mRNA electroporated CAR T cells were loaded with indo-1 and stimulatedwith mesothelin-Fc fusion protein. Calcium influx in cells was measuredpost stimulation by observing the ratio of bound to free calcium.

The results of the experiments are now described.

Results

Normal human donor CD4 and CD8 T cells were obtained and stimulated witha combination of αCD2 (T11.1 and T11.2), αCD3 (OKT3), and/or αCD28 (9.3)antibody coated magnetic beads. After stimulation, cell proliferationwas assessed by observing the absolute cell number and populationdoublings, while cell activation was assessed by measuring cell size.Activation by CD2/CD3 signals sufficiently induces cell activation andcell proliferation of both CD4 and CD8 T cell populations (FIG. 2).

Human T cells were electroporated with mRNA for various CAR constructs,either containing or not containing the CD2 signaling domain. The mRNAelectroporated T cells were co-cultured for 24 hours with target tumorcells after which cytokine secretion was measured. IL-2 and IFN-γsecretion is increased in SS1-CD2z, similar to what is observed inSS1-28z. However, IL-2 and IFN-γ secretion is diminished in SS1-28CD2z.Similarly, anti-CD19-CD2z produces similar IL-2 and IFN-γ compared toanti-CD19-z and anti-CD19-28z, but IL-2 and IFN-γ secretion isdiminished in anti-CD19-28CD2z CARs (FIG. 3). The observation that CD2signaling domains allow CAR T cells to proliferate while diminishingcytokine secretion may be important in some therapeutic instances. Forinstance, in one case a patient with leukemia treated with a CD19 CARcontaining a CD28 signaling domain succumbed due to high levels ofcytokines (Brentjens et al, 2010, Mol Ther, 8(4):666-8). Similarly, apatient with colon cancer died shortly after being injected withher2/neu CAR T cells that contained a CD28 signaling domain (Morgan etal, 2010, Mol Ther, 18(4):843-51).

To assess the tumor killing ability of CD2-containing CARs, mRNAelectroporated T cells were co-cultured with target tumor cells atvarying effector to target ratios. After 4 hours of co-culture, anti-CD19-CD2z CARs efficiently eliminated K562-CD19 tumor cells, whileSS1-CD2z CARs efficiently eliminated K562-meso tumor cells. Further,after 18 hours of co-culture, SS1-CD2z displayed effective killing ofK562-meso tumor cells (FIG. 4).

To evaluated calcium signals, mRNA CAR electroporated T cells wereloaded with Indo-1AM calcium indicator and stimulated with mesothelin-Fcfusion protein (30 seconds after recording). The ratio of bound to freecalcium was recorded over time. Inclusion of the CD2 signaling domainsquelches calcium flux, as can be seen by the reduction in calciuminflux in SS1-28CD2z compared to SS1-28z (without CD2) (FIG. 5). Whilenot wishing to be held to any particular theory, the observation thatCD2 signaling diminishes the calcium flux is likely causally related tothe diminished cytokine secretion. This may be important in sometherapeutic instances. For instance, in one case a patient with leukemiatreated with a CD19 CAR containing a CD28 signaling domain succumbed dueto high levels of cytokines (Brentjens et al, 2010, Mol Ther,8(4):666-8). Similarly, a patient with colon cancer died shortly afterbeing injected with her2/neu CAR T cells that contained a CD28 signalingdomain (Morgan et al, 2010, Mol Ther, 18(4):843-51).

The results presented herein show that CD2z CARs induce cytokineproduction on par with 28z CARs, demonstrate a proliferation advantageover first generation CARs (z-alone), and exhibit a calcium fluxresponse intermediate between what was observed for 28z and BBz (FIG.6).

It is herein reported that use of the CD2 signaling domain insecond-generation CARs influences IL-2 and IFN-γ production of CAR-Tcells and exhibits similar target cell lysis. These results demonstratethat CD2 can be used in second-generation CAR molecules to alter thecytokine production of CAR-T cells in both negative and positivedirections and may have implications for sustainability andeffectiveness of CAR-T cells in tumor microenvironments in vivo byaltering CAR T cell survival and activation induced cell deaththresholds. Given the high self-activation of the SS-128z CAR, SS1-CD2zmay be a more tolerable CAR for in vivo mesothelin tumor attack and maydecrease activation induced cell death.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. An isolated nucleic acid sequence encoding achimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain, a transmembrane domain, a costimulatory signalingregion, and a CD3 zeta signaling domain, wherein the costimulatorysignaling region comprises the CD2 signaling domain.
 2. The isolatednucleic acid sequence of claim 1, comprising the nucleic acid sequenceof SEQ ID NO:
 1. 3. The isolated nucleic acid sequence of claim 1,wherein the antigen binding domain is an antibody or an antigen-bindingfragment thereof.
 4. The isolated nucleic acid sequence of claim 1,wherein the antigen binding domain binds to a tumor antigen.
 5. Theisolated nucleic acid sequence of claim 1, wherein the costimulatorysignaling region further comprises the intracellular domain of acostimulatory molecule selected from the group consisting of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, a ligand that specificallybinds with CD83, and any combination thereof.
 6. An isolated chimericantigen receptor (CAR) comprising an antigen binding domain, atransmembrane domain, a costimulatory signaling region, and a CD3 zetasignaling domain, wherein the costimulatory signaling region comprisesthe CD2 signaling domain
 7. The isolated CAR of claim 6, wherein theantigen binding domain is an antibody or an antigen-binding fragmentthereof.
 8. The isolated CAR of claim 6, wherein the antigen bindingdomain binds to a tumor antigen.
 9. The isolated CAR of claim 6, whereinthe costimulatory signaling region further comprises the intracellulardomain of a costimulatory molecule selected from the group consisting ofCD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, and any combination thereof.10. A cell comprising a nucleic acid sequence encoding a chimericantigen receptor (CAR), the CAR comprising an antigen binding domain, atransmembrane domain, a costimulatory signaling region, and a CD3 zetasignaling domain, wherein the costimulatory signaling domain comprisesthe CD2 signaling domain.
 11. The cell of claim 10, wherein the nucleicacid comprises the nucleic acid sequence of SEQ ID NO:
 1. 12. The cellof claim 10, wherein the antigen binding domain is an antibody or anantigen-binding fragment thereof.
 13. The cell of claim 10, wherein theantigen binding domain binds to a tumor antigen.
 14. The cell of claim10, wherein the costimulatory signaling region further comprises theintracellular domain of a costimulatory molecule selected from the groupconsisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C,B7-H3, a ligand that specifically binds with CD83, and any combinationthereof.
 15. The cell of claim 14, wherein the cell is selected from thegroup consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic Tlymphocyte (CTL), and a regulatory T cell.
 16. The cell of claim 10,wherein the cell exhibits an anti-tumor immunity when the antigenbinding domain binds to its corresponding antigen.
 17. A vectorcomprising a nucleic acid sequence encoding a chimeric antigen receptor(CAR), wherein the CAR comprises an antigen binding domain, atransmembrane domain, a costimulatory signaling region, and a CD3 zetasignaling domain, wherein the costimulatory signaling region comprisesthe CD2 signaling domain.
 18. The vector of claim 17, wherein theisolated nucleic acid sequence encoding a CAR comprises the nucleic acidsequence of SEQ ID NO:
 1. 19. The vector of claim 17, wherein theantigen binding domain is an antibody or an antigen-binding fragmentthereof.
 20. The vector of claim 17, wherein the antigen binding domainbinds to a tumor antigen.
 21. The vector of claim 17, wherein thecostimulatory signaling region further comprises the intracellulardomain of a costimulatory molecule selected from the group consisting ofCD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, and any combination thereof.22. A method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal, the method comprisingadministering to a mammal an effective amount of a cell geneticallymodified to express a CAR, wherein the CAR comprises an antigen bindingdomain, a transmembrane domain, a costimulatory signaling regioncomprising the CD2 signaling domain, and a CD3 zeta signaling domain,and wherein the antigen binding domain is selected to specificallyrecognize the target cell population or tissue.
 23. A method ofproviding an anti-tumor immunity in a mammal, the method comprisingadministering to the mammal an effective amount of a cell geneticallymodified to express a CAR, wherein the CAR comprises an antigen bindingdomain, a transmembrane domain, a costimulatory signaling regioncomprising the CD2 signaling domain, and a CD3 zeta signaling, therebyproviding an anti-tumor immunity in the mammal.
 24. A method of treatinga mammal having a disease, disorder or condition associated with anelevated expression of a tumor antigen, the method comprisingadministering to the mammal an effective amount of a cell geneticallymodified to express a CAR, wherein the CAR comprises an antigen bindingdomain a transmembrane domain, a costimulatory signaling regioncomprising the CD2 signaling domain, and a CD3 zeta signaling domain,thereby treating the mammal.
 25. The method of claim 24, wherein thecell is an autologous T cell.
 26. The method of claim 24, wherein thetumor antigen is selected from the group consisting of CD19, CD20, CD22,ROR1, mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII,GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and any combination thereof.
 27. Amethod of treating a human with cancer, the method comprisingadministering to the human a T cell genetically engineered to express aCAR, wherein the CAR comprises an antigen binding domain, atransmembrane domain, a costimulatory signaling region comprising theCD2 signaling domain, and a CD3 zeta signaling domain.
 28. The method ofclaim 27, wherein the human is resistant to at least onechemotherapeutic agent.
 29. A method of generating a persistingpopulation of genetically engineered T cells in a human diagnosed withcancer, the method comprising administering to the human a T cellgenetically engineered to express a CAR, wherein the CAR comprises anantigen binding domain a transmembrane domain, a costimulatory signalingregion comprising the CD2 signaling domain, and a CD3 zeta signalingdomain, wherein the persisting population of genetically engineered Tcells persists in the human for at least one month after administration.30. The method of claim 29, wherein the persisting population ofgenetically engineered T cells comprises at least one cell selected fromthe group consisting of a T cell that was administered to the human, aprogeny of a T cell that was administered to the human, and acombination thereof.
 31. The method of claim 29, wherein the persistingpopulation of genetically engineered T cells comprises a memory T cell.32. The method of claim 29, wherein the persisting population ofgenetically engineered T cells persists in the human for at least threemonths after administration.
 33. The method of claim 29, wherein thepersisting population of genetically engineered T cells persists in thehuman for at least four months, five months, six months, seven months,eight months, nine months, ten months, eleven months, twelve months, twoyears, or three years after administration.
 34. The method of claim 29,wherein the cancer is treated.
 35. A method of expanding a population ofgenetically engineered T cells in a human diagnosed with cancer, themethod comprising administering to the human a T cell geneticallyengineered to express a CAR, wherein the CAR comprises an antigenbinding domain, a transmembrane domain, a costimulatory signaling regioncomprising the CD2 signaling domain, and a CD3 zeta signaling domain,wherein the administered genetically engineered T cell produces apopulation of progeny T cells in the human.
 36. The method of claim 35,wherein the progeny T cells in the human comprise a memory T cell. 37.The method of claim 35, wherein the T cell is an autologous T cell. 38.The method of claim 35, wherein the population of progeny T cellspersists in the human for at least three months after administration.39. The method of claim 35, wherein the population of progeny T cellspersist in the human for at least four months, five months, six months,seven months, eight months, nine months, ten months, eleven months,twelve months, two years, or three years after administration.
 40. Amethod of modulating the amount of cytokine secreted by a T cell, saidmethod comprising genetically engineering the T cell to express a CAR,wherein the CAR comprises an antigen binding domain, a transmembranedomain, a costimulatory domain region comprising CD2, and a CD3 zetasignaling domain.
 41. The method of claim 40, wherein modulating theamount of cytokine secreted by a T cell reduces the proliferation of Tregulatory cells.
 42. A method of reducing the amount ofactivation-induced calcium influx into a T cell, said method comprisinggenetically engineering the T cell to express a CAR, wherein the CARcomprises an antigen binding domain, a transmembrane domain, acostimulatory domain region comprising CD2, and a CD3 zeta signalingdomain.
 43. The method of claim 42, wherein reducing the amount ofactivation-induced calcium influx into a T cell prevents theactivation-induced cell death of the T cell.